Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking.
Direct mapping of an agonist-binding domain within the parathyroid hormone/parathyroid hormone-related protein receptor by photoaffinity crosslinking.
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通过光亲和交联直接绘制甲状旁腺激素/甲状旁腺激素相关蛋白受体内的激动剂结合域。
DOI：
10.1073/pnas.94.8.3644
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发表时间：
1997
影响因子：
11.1
通讯作者：
Chorev,M
中科院分区：
文献类型：
--
作者：
Zhou,AT;Bessalle,R;Bisello,A;Nakamoto,C;Rosenblatt,M;Suva,LJ;Chorev,M
甲状旁腺激素（PTH）和PTH相关蛋白（PTHrP）是一种与共同的七跨膜结构域G蛋白偶联受体相互作用的促钙激素，主要存在于骨和肾中。为了绘制激素与受体之间的双分子相互作用界面，我们设计了一种具有生物活性、光反应性的PTH激动剂125I-[Nle8,18,Lys13(] -p-(3-I-Bz)Bz),l-2-Nal23,Arg26,27,Tyr34]bPTH-(1-34)NH2（125I-all- r - k13）。该配体含有一个光反应性的二苯甲酮片段，连接在Lys13的侧链上。所有其他赖氨酸残基都被银基取代。在人胚胎肾细胞（HEK-293/C-21细胞，每个细胞约400,000个受体）中，该类似物与重组hPTH/PTHrP受体特异性光交联，在SDS/PAGE放射自显影上产生约87-kDa的弥漫性条带。为了确定参与125i -all- r - k13结合的hPTH/PTHrP受体内的“接触域”，对含有配体-受体偶联物的放射性标记带进行了化学和酶切。采用两种独立的顺序消化途径：途径A，赖氨酸内肽酶C，内切n -糖苷酶F，溴化氰；途径B是溴化氰，然后是内切n -糖基酶f。鉴定的结构域与125i -all- r - k13中的13位接触，对应于位于n端胞外结构域c端区域的hPTH/PTHrP受体的173-189氨基酸。
Parathyroid hormone (PTH) and PTH-related protein (PTHrP) are calciotropic hormones interacting with a shared seven-transmembrane domain G protein-coupled receptor, which is located predominantly in bone and kidney. To map the interface of the bimolecular interaction between hormone and receptor, we designed and radioiodinated a bioactive, photoreactive PTH agonist,125I-[Nle8,18,Lys13(ɛ-p-(3-I-Bz)Bz),l-2-Nal23,Arg26,27,Tyr34]bPTH-(1–34)NH2(125I-all-R-K13). This ligand contains a photoreactive benzophenone moiety attached to the side chain of Lys13. All other lysyl residues are substituted by argynyls. The analog photocrosslinks specifically to the recombinant hPTH/PTHrP receptor stably transfected into human embryonic kidney cells (HEK-293/C-21 cells, ≈400,000 receptors per cell), generating a diffuse ≈87-kDa band on SDS/PAGE autoradiography. To identify the “contact domain” within the hPTH/PTHrP receptor involved in binding of125I-all-R-K13, the radiolabeled band containing the ligand–receptor conjugate was subjected to chemical and enzymatic cleavage. Two independent pathways of sequential digestion were used: Route A, lysyl endopeptidase C, then endo-N-glycosidase F, followed by cyanogen bromide; Route B, cyanogen bromide followed by endo-N-glycosydase F. The identified domain is in contact with position 13 in125I-all-R-K13 and corresponds to amino acids 173–189 of the hPTH/PTHrP receptor, located at the C-terminal region of the N-terminal extracellular domain.